Signal transduction pathways are used by all cells to detect environmental stimuli and generate appropriate behavioral responses. These pathways are critical components of human disease, allowing pathogens to detect and invade the human host and when not functioning properly, leading to cancer and other diseases. In both prokaryotic and eukaryotic organisms, signal transduction pathways involve multiple protein-protein interactions that facilitate information transfer by covalent modification of proteins (e.g. phosphorylation) or interaction-mediated conformational changes in the proteins. Understanding protein-protein interactions within signal transduction pathways will benefit a wide range of fields impinging on human health. The chemotaxis system of bacteria represents the best studied signal transduction in biology today. This pathway allows bacteria to detect external stimuli via chemoreceptors in the cell membrane and control the cell's swimming behavior through phosphorylation of a response regulator protein by a histidine protein kinase. The chemotaxis pathway has been studied for many years, providing a wealth of structural, biochemical, and genetic information. However, many important questions about the system remain unanswered: how the signaling complex is assembled, for example: how signals are terminated, and how covalent modification of receptors contributes to adaptation. Our long term goal is to understand how living cells detect, transmit, and adapt to various signals on a molecular level. In this proposal we will apply computational genomic and biophysical approaches to understanding three key steps of the bacterial chemotaxis signal transduction pathway: excitation, signal termination, and adaptation. Novel methodology will be utilized to study protein- protein interactions in each of these areas. This will involve creating a natural classification of chemotaxis proteins based on phylogenetic analysis, identification of conserved residues within evolutionarily related subgroups, co-variance analysis of co-evolving residues, and molecular docking simulations to test models of protein-protein interactions. In Aim 1, these methods will be applied to the interactions between the chemoreceptors (MCPs), the scaffolding protein (CheW), and the histidine kinase (CheA) involved in the excitation pathway. Aim 2 will concentrate on interactions between the response regulator (CheY) and phosphatases involved in termination of the excitatory signal. Aim 3 will investigate interactions critical to the adaptation pathway involving covalent modification of the chemoreceptor by enzymes with methytransferase, methylesterase, or deamidase activities. This work will produce testable models of protein-protein interactions within the bacterial chemotaxis pathway that will drive further experimental and systems biology research by our collaborators and other laboratories. The principles learned through these studies will provide important information about signal transduction and aid the design of new therapeutics targeting the signaling pathways that control virulence in human pathogens.